Running the protocol

The Phosphopeptide Enrichment protocol:

•	Washes the syringes.

•	Primes and equilibrates the cartridges to prepare for sample loading.

•	Loads the samples onto the cartridges.

•	Removes non-specific binding molecules from the cartridges.

•	Elutes the peptides from the cartridges.

For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the cartridge seating station.

Before you start

Ensure that you:

•	Prepare the reagents. See Preparing the reagents.

•	Prepare the samples. See Preparing the samples.

•	Run the Startup protocol to prepare the AssayMAP Bravo Platform for the run. See the System Startup/Shutdown v2.2 utility.

•	Transfer the cartridges to the 96AM Cartridge & Tip Seating Station. See Cartridge Transfer v1.1.

 

Cartridges ship dry and, therefore, contain air entrained in the cartridge bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

 

 

Do not allow wetted cartridges to dry out.

 

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Phosphopeptide Enrichment application.

To set up the protocol:

1	Open the App library. See Accessing an application.

2	Locate Phosphopeptide Enrichment, and then click App.

 

The Phosphopeptide Enrichment application opens.

 

3	Optional. Click Restore Defaults to set all Application Settings to their defaults.

4	In the Application Settings area, specify the following:

 

If the column selection is greater than the actual number of cartridges used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 10 columns, but only 1 column of cartridges are in the seating station, the head will apply 10 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

 

 

If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 10 columns of cartridges are in the seating station, the head will apply 1/10th the force required to seat the cartridges properly. To prevent potential equipment damage, ensure that the column selection is correct.

 

 

Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

 

a	In the Application Settings area, specify the Number of Full Columns of Cartridges to use during the run. The position of the columns of cartridge in the tip seating station must match the positions of the samples and solutions in the plates on the deck.

•	Default: 1

•	Range: 1–12

b	Select the check boxes of the Steps that you want to perform.

c	Enter values for the selected Steps.

See the Application Settings table for the default and the practical range of values for each step.

5	In the Labware Table area, select the labware that you are using for the protocol run.

 

Incorrect labware selections can cause a hardware collision, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck.

 

6	Optional. When you are finished, click Save Settings.

 

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Application Settings table

The following table gives a brief description of each setting. For more details, including the practical and full ranges of values for a given setting, see the Assay development guidelines and protocol notes.

 

 

Steps	Description
Initial Syringe Wash	Rinses syringes with deionized water using the specified number of cycles at the wash station. Wash Cycles: Default: 3
Prime	Defines the volume of Priming Buffer, the flow rate used to Prime the cartridges, and the number of syringe wash cycles at the wash station. Volume (µL): Default: 100 Flow Rate (µL/min): Default: 300 Wash Cycles: Default: 1
Equilibrate	Defines the volume of Equilibration Buffer, the flow rate used to equilibrate the cartridge, and the number of syringe wash cycles at the wash station. Volume (µL): Default: 50 Flow Rate (µL/min): Default: 10 Wash Cycles: Default: 1
Load Samples	Defines the volume of sample, the flow rate used to load samples, and the number of syringe wash cycles at the wash station. IMPORTANT Ensure that each sample well has the recommended overage (labware dependent) to prevent air from getting introduced into the cartridge. Volume (µL): Default: 100 Flow Rate (µL/min): Default: 5 Wash Cycles: Default: 3
Collect Flow Through (Load Samples step)	If selected, dispenses the Load Samples flow-through to the Flow Through Collection plate. If unselected, the flow-through is dispensed into the Organic Waste plate.
Cup Wash	Defines the volume of Cartridge Wash Buffer used to wash out each cartridge cup and the number of cycles used to perform a cup wash with an accompanying syringe wash at the wash station. Volume (µL): Default: 25 Wash Cycles: Default: 1
Internal Cartridge Wash	Defines the volume of Cartridge Wash Buffer, the flow rate used to wash non-target elements from the cartridge resin, and the number of syringe wash cycles at the wash station. A 5-µL sample chase with Cartridge Wash Buffer occurs at the Load Samples flow rate before the Internal Cartridge Wash step. Volume (µL): Default: 50 Flow Rate (µL/min): Default: 10 Wash Cycles: Default: 3
Collect Flow Through	If selected, collects the Internal Cartridge Wash flow-through at the Flow Through Collection plate. If not selected, discards the Internal Cartridge Wash flow-through into the Organic Waste plate.
Stringent Syringe Wash	Defines the volume of Syringe Wash Buffer and the number of cycles used to perform the Stringent Syringe Wash step with an accompanying syringe wash at the wash station. Volume (µL): Default: 50 Wash Cycles: Default: 1
Elute	Defines the volume of Elution Buffer, the flow rate used to elute analytes from the cartridges, and the number of syringe wash cycles at the wash station. Volume (µL): Default: 20 Flow Rate (µL/min): Default: 5 Wash Cycles: Default: 1
Eluate Discard	If selected, a specified initial volume of the Eluate will be collected at the Flow Through Collection plate, or discarded at the Organic Waste plate. Volume (µL): Default: 0
Add to Flow Through	If selected, collects the Eluate Discard at the Flow Through Collection plate. If not selected, discards the Eluate Discard into the Organic Waste plate.
Existing Collection Volume	Defines the volume of buffer in the Eluate Collection plate before the start of the protocol. This value is used to calculate the sample mixing volume and to adjust the well-bottom offset for elution. Volume (µL): Default: 0
Final Syringe Wash	Washes the syringes the specified number of cycles at the wash station. Wash Cycles: Default: 3

 

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles.

You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 µL/min and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

 

The protocol will not run if cartridges are missing. You can use resin-free cartridges instead of packed cartridges for mock runs.

 

 

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Starting the protocol run

To start the protocol run:

1	Place the filled labware on the deck at the positions indicated in the Deck Layout area.

 

The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

 

2	Make sure the labware is properly seated on the Bravo deck.

 

Improperly seated labware can cause a hardware collision, resulting in equipment damage. Ensure that all labware are properly seated within the alignment features of their respective platepads.

 

3	Review the selections and value specifications in the Phosphopeptide Enrichment protocol to confirm they are correct. Make sure:

•	The physical layout on the AssayMAP Bravo deck matches the display in the Deck Layout area.

•	In the Labware Table area, the selections reflect the labware you are using for the protocol run.

 

Incorrect labware selections can cause a hardware collision, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck.

 

4	Click Run Protocol.

An error message appears if the software detects an error in the form settings, for example, if an entered value is outside the limits set for a field. If this happens, follow the directions in the message to select Pause and Diagnose, and then select Abort process. After setting the appropriate values, click Run Protocol to restart the run.

Note: For the range of acceptable values for a given setting, see the Assay development guidelines and protocol notes.

To monitor the progress of the run, check the Status box.

 

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol. The default protocol with 100‑µL samples and 5-µL/min sample loading flow rate should take approximately 50 minutes to complete.

 

To stop a run in an emergency, use the hardware Emergency Stop button.

 

 

Protocols should not be paused to change the settings. However, if you need to change a setting in a running protocol, click Pause, and then click Abort process in the dialog box that appears. Change the setting, and then clear the check boxes of the steps in the Phosphopeptide Enrichment application that have been completed successfully. Restart the protocol to complete the remaining steps.

 

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses.

To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

 

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Cleaning up

When the protocol run is finished, make sure you:

•	Remove the labware from the AssayMAP Bravo deck.

•	Discard the liquid from the Organic Waste plate.

Because the Phosphopeptide Enrichment protocol uses purified, deionized water in the source bottle for the wash station, no additional special cleanup procedures are required for the AssayMAP Bravo Platform.

•	Run the Shutdown utility. See System Startup/Shutdown v2.2.

You should run the Shutdown utility after every application protocol run, or if the AssayMAP Bravo Platform will be idle for longer than 1 hour.

 

Make sure you discard the chemical waste and used labware according to your lab’s waste disposal procedures and in compliance with all local, state, and federal safety regulations.

 

 

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